Chromosome segregation is essential for the propagation of species and the viability of cells, and is driven by a complex microtubule-based superstructure called the spindle. Cumulative evidence demonstrates that spindle organization and chromosome movement are driven by the concerted actions of microtubule-associated proteins (motor and non-motor, structural proteins) and the inherent dynamic properties of microtubules. Our goal is to use in vitro biochemical techniques to identify proteins essential for spindle organization and then to apply in vitro and in vivo cell biological techniques to determine how those proteins contribute to spindle morphogenesis and chromosome movement. Specifically, we will continue to exploit a cell free assay for spindle pole organization that we developed previously. We propose to use it as an enriched source for purification of enzymes that regulate spindle formation and as a source of microtubule asters for biophysical analyses. We also propose to use live cell imaging to define how specific proteins and protein complexes contribute to both spindle morphogenesis and chromosome movement in mitosis. These combined approaches will generate insight into the molecular mechanisms of spindle assembly and chromosome movement in mammalian cells. The specific aims of this research are to: 1) identify specific sites of mitosis-specific phosphorylation on the spindle organizing protein NuMA and use in vitro and in vivo assays to determine how those modifications regulate NuMA function; 2) use chromatographic techniques to isolate and identify enzymes the regulate NuMA function during mitosis; 3) use live cell imaging to determine how bipolar spindles organize in the absence of two KinI kinesin proteins; 4) combine live cell microscopy with RNAi knock down to determine how kinetochores elaborate spindle microtubules; and 5) use optical trapping microscopy to directly measure force on microtubule minus ends at spindle poles.